rabbit polyclonal antibodies against flag tag  (Proteintech)

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a Distribution of fibroblast subtypes expressed as a difference between FD-treated and FD-fellow sclera (two-sided chi-square test with Bonferroni correction for multiple testing). The X-axis indicates the subtypes corresponding to the uniform manifold approximation and projection plot. The Y-axis shows the log-transformed P -value obtained by the chi-square test between FD-treated and FD-fellow eyes’ scleral fibroblast subtypes. Positive values indicate a decrease, whereas negative values indicate an increase in the FD-treated eyes when compared with the FD-fellow eyes. b Heat map of the ECM outgoing (left) and incoming (right) of cell-cell interaction in all fibroblast subtypes. c Heat map of the outgoing (left) and incoming (right) cell-cell interaction signals of all fibroblast subtypes. The row indicates the relative strength of each interaction pathway. The column indicates the total interaction strength of each fibroblast subtype. d Ligand-receptor pairs of the ncWNT signalling pathway of all fibroblast subtypes. The relative contribution of each ligand-receptor pair of the ncWNT signalling pathway among all scleral fibroblast subtypes. e Expression profile of Wnt5a of ncWNT signalling in each subtype of fibroblast. f Expression profile of Wnt11 of ncWNT signalling in each fibroblast subtype. g Wnt5a mRNA level in FD-48h treated eyes and FD-48h fellow eyes (two-sided Wilcoxon signed-rank test). h Information flow of each cell–cell interaction pathway in FD treatment for 24 h or 48 h. FD form deprivation. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Transformation Assay, Expressing

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a Locations of regionally differentiated sclera: iPPS, oPPS, and peripheral sclera. b Alteration of the number and percentage of Wnt5a + A2m + cells and Wnt5a + puncta in the temporal and nasal iPPS, oPPS, and peripheral sclera in normal murine eyes at age 3 weeks. n = 18 micrographs; Friedman test with Dunn’s post hoc test (two-sided) or RM-ANOVA with Bonferroni’s post hoc test (two-sided). c , d The numbers and percentages of Wnt5a + A2m + cells and Wnt5a + puncta in the temporal/nasal iPPS, oPPS, and peripheral sclera in NC eyes, FD-F eyes, and FD-T eyes. n = 9 micrographs for NC eyes; for FD eyes, n = 12 micrographs in FD-24h and n = 9 micrographs in FD-48h, one-way ANOVA with Bonferroni’s post hoc test or Kruskal-Wallis non-parametric test with Dunn’s post hoc test (two-sided). e The Wnt5a mRNA level in NC eyes, FD-F eyes, and FD-T eyes treated by FD-48 h or FD-2W. For NC eyes, n = 10 in FD-48 h and n = 18 in FD-2W; for FD-F eyes and FD-T eyes, n = 9; one-way ANOVA with Bonferroni’s post hoc test. WNT5A protein immunofluorescence intensity ( f ) and distribution ( g ) in the temporal/nasal iPPS, oPPS, and peripheral sclera in normal murine eyes. n = 11, Friedman tests with Dunn’s post hoc tests (two-sided). WNT5A protein immunofluorescent intensity ( h ) and change ( i ) in the temporal/nasal iPPS, oPPS, and peripheral sclera in NC eyes, FD-F eyes, and FD-T eyes. For NC eyes, n = 5 in FD-48 h and n = 6 in FD-2W; for FD-F eyes and FD-T eyes, n = 5, one-way ANOVA with Bonferroni’s post hoc test or Kruskal–Wallis non-parametric test with Dunn’s post hoc test (two-sided). Western blots ( j ) and densitometric quantification ( k ) of scleral WNT5A protein in FDM mice. n = 6, one-way ANOVA with Bonferroni’s post hoc test (two-sided). red: Wnt5a ; green: A2m ; blue: 4’, 6-diamidino-2-phenylindole (DAPI), iPPS the inner peripapillary sclera, oPPS the outer peripapillary sclera, peripheral the peripheral sclera. NC normal control eyes, FD-F untreated contralateral eyes of form deprivation mice, FD-T form-deprived eyes. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Immunofluorescence, Western Blot, Control

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a sh Wnt5a -AAV injection and refraction and ocular biometrics measurements were carried out at different time points. b AAV infection in the whole murine sclera after sh Wnt5a sub-Tenon’s injection. c The scleral Wnt5a mRNA level declined by sh Wnt5a -AAV injection. n = 6 for sh Scramble -AAV injected group and n = 7 for sh Wnt5a -AAV injected group, Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc test (two-sided). d , e WNT5A protein levels were significantly downregulated by sh Wnt5a -AAV injection. n = 7 mice per group, Friedman test with Dunn’s post hoc test (two-sided). f The refraction of sh Wnt5a -AAV injected eyes underwent a greater myopic shift than did the shScramble-AAV injected mice. n = 15 for the shScramble-AAV injected group and n = 13 for the sh Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc test. g Compared with shScramble-AAV infection, sh Wnt5a -AAV infection caused a significant increase in axial length. n = 15 for the shScramble-AAV injected group and n = 13 for the sh Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc test. Col1a1 and Acta2 mRNA ( h , for Col1a1 , n = 6 for shScramble-AAV injected group and n = 10 for sh Wnt5a -AAV injected group, Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc test, two-sided; for Acta2 , n = 7 for shScramble-AAV injected group and n = 9 for sh Wnt5a -AAV injected group, one-way ANOVA with Bonferroni’s post hoc test, two-sided) and protein ( i , j, n = 7 for COL1A1 and n = 3 for α-SMA, RM-ANOVA with Bonferroni’s post hoc test (two-sided) for COL1A1 and Friedman test with Dunn’s post hoc test (two-sided) for α-SMA) level in shScramble-AAV, sh Wnt5a -AAV injected mice. k scleral fibril ultrastructure and scleral fibril diameter of the fellow eyes and the treated eyes in shScramble-AAV injected mice and sh Wnt5a -AAV injected mice ( n = 3 mice per group). Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Injection, Infection

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: Six weeks after sub-Tenon injection of OE Wnt5a -AAV, Wnt5a was overexpressed in the sclera at mRNA ( a , n = 5 for vector -AAV injected group and n = 9 for OE Wnt5a -AAV injected group, Kruskal–Wallis non-parametric test with Dunn’s post hoc test) and protein levels ( b , c, n = 3, RM-ANOVA with Bonferroni’s post hoc test, two-sided). d Schematic schedule of FD treatment and Wnt5a overexpression with AAV injection. In the FDM mouse model, OE Wnt5a -AAV injected + FDM mice developed less myopic shift than did the vector-AAV injected mice ( e ). In comparison with the vector-AAV injected eyes, OE Wnt5a -AAV injected + FD eyes exhibited a significant difference for axial length ( f, n = 13 for vector-AAV injected group and n = 16 for OE Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc tests, two-sided, for refraction and for axial length). g , h Wnt5a overexpression can partially rescue the COL1A1 protein level, which is downregulated by FD. n = 7, ordinary two-way ANOVA with Bonferroni’s post hoc test (two-sided). In mice undergoing normal refractive development ( i ), sub-Tenon’s injection of OE Wnt5a -AAV did not affect refraction ( j ) or axial length ( k ). n = 11 for the vector-AAV injected group and n = 15 for the OE Wnt5a -AAV injected group, two-way repeated measures ANOVA with Bonferroni’s post hoc test (two-sided). FD form deprivation; FDM form deprivation myopia. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Injection, Plasmid Preparation, Over Expression, Comparison

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: a , b Gene ontology (GO) and Reactome pathway analysis of differentially expressed genes (DEGs) from scleral bulk RNA-seq after sub-Tenon’s injection of sh Wnt5a -AAV ( n = 2 each group, one-sided hyper-geometric test with Benjamini-Hochberg correction for multiple testing). c DEGs heatmap of the ECM pathway from scleral bulk RNA-seq analysis. d Reverse Transcription quantitative Polymerase Chain Reaction validation of the DEGs ( Sparc , Adamts2 , Pcolce2 , Pcolce , Bmp1 , Itga2 ) from bulk RNA-seq analysis. For Sparc, n = 4 for shScramble-AAV injected group and n = 6 for sh Wnt5a -AAV injected group; for Adamts2 , Pcolce2 and Bmp1, n = 5 for both shScramble-AAV injected group and sh Wnt5a -AAV injected group; for Pcolce, n = 3 for shScramble-AAV injected group and n = 5 for sh Wnt5a -AAV injected group; for Itga2 , n = 4 for shScramble-AAV injected group and n = 5 for sh Wnt5a -AAV injected group. One-way ANOVA with Bonferroni’s post hoc test (two-sided) for Sparc, Adamts2 , and Pcolce , Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc tests (two-sided) for Pcolce2, Bmp1 , and Itga2 . e , f Network of the 20 hub genes and functional enrichment (one-sided hyper-geometric test with Benjamini–Hochberg correction for multiple testing) of the Wnt5a assigned gene module. g Western blots and densitometric quantification of scleral WNT5A and SAPRC proteins; representative blots are shown for sh Wnt5a -AAV injected mice. n = 5, Friedman tests with Dunn’s post hoc test (two-sided) for WNT5A; RM-ANOVA with Bonferroni’s post hoc test (two-sided) for SAPRC. h , i Immunofluorescence intensity and quantification of SPARC protein expression in the temporal/nasal iPPS, oPPS, and peripheral sclera of NC eyes. In FD-48h, n = 6 mic per group; in FD-2W, n = 3 for NC eyes and n = 4 for both FD-F and FD-T eyes. One-way ANOVA with Bonferroni’s post hoc test (two-sided), Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 post hoc test (two-sided) or Kruskal–Wallis non-parametric test with Dunn’s post hoc test (two-sided) were used. Red: SPARC; blue: 4’, 6-diamidino-2-phenylindole (DAPI); iPPS the inner peripapillary sclera, oPPS the outer peripapillary sclera, peripheral, the peripheral sclera. NC normal control eyes, FD-F untreated contralateral eyes of form deprivation mice, FD-T form deprivation treated eyes. Data are expressed as mean ± SEM.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: RNA Sequencing, Injection, Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Functional Assay, Western Blot, Immunofluorescence, Expressing, Control

Journal: Nature Communications

Article Title: Decreased scleral Wnt5a hi fibroblasts exacerbate myopia progression by disrupting extracellular matrix homeostasis in mice

doi: 10.1038/s41467-025-67246-x

Figure Lengend Snippet: Abnormal visual stimulation propagating via the retina-choroid-sclera signalling cascade reduces Wnt5a hi fibroblast numbers and/or activity, and thus Wnt5a secretion. Subsequently, the expression of genes for ECM molecules such as Col1a1 , Sparc , Pcolce , Pcelce2 , Adamts2 , Bmp1 , and Igta2 is downregulated via the noncanonical Wnt signalling pathway (Wnt5a-Ca 2+ -CaMKII). This response may disrupt ECM structural organisation, decrease collagen I level, and reduce the collagen fibril diameter, finally resulting in myopia progression due to increased distension of the posterior sclera under constant intraocular pressure.

Article Snippet: The sections were blocked with 6% normal donkey serum, 1% BSA, and 0.2% Triton X-100 in 0.1 M PBS for 2 h at room temperature, then incubated with rabbit polyclonal antibodies against WNT5A (1:100, #bs-1948R, Bioss, Woburn, MA, USA) and SPARC (1:300, #15274-1-AP, Proteintech, Rosemont, IL, USA) for 24 h at 4 °C.

Techniques: Activity Assay, Expressing